Molecular biology methods have allowed researchers to investigate the genetic basis of disease processes and identify molecular events, such as aberrant genetic splicing, deletions, insertions, substitutions and amplifications responsible for genetic defects associated with some diseases, such as cancer. Certain aberrant genetic splicing events are nonrandom and characteristic of particular diseases.
In eukaryotes, the genetic information in DNA is present in chromosomes contained within a nucleus. Some viruses also contain chromosome-like genetic structures. The genetic information contained within one strand of DNA depends on the sequence of bases (i.e., “bass sequence” or “nucleotide sequence”) where the bases are adenine (A), guanine (G), cytosine (C) and thymine (T). DNA encodes complementary messenger ribonucleic acid (mRNA) sequences, in which T is replaced with uracil (U) and the 5′ to 3′ nucleotide sequence specifies the amino acid sequence of the encoded protein. Eukaryotic genes generally include noncontiguous coding regions (“exons”) separated by intervening non-coding regions (“introns”). Nuclear transcription synthesizes a precursor mRNA (pre-mRNA) complementary to the coding DNA strand, including introns and exons. Pro-mRNA is processed to eliminate the introns by cleaving and splicing the RNA to covalently link the exons and concurrently excise the introns. The resulting mature mRNA exits the nucleus into the cytoplasm where translation occurs during protein synthesis.
Nucleic acid splicing also occurs in the life cycle of many viruses. Some viruses integrate their nucleic acid into a host cell's DNA as a provirus using a splicing mechanism in which the host DNA is cleaved followed by insertion and ligation of the viral nucleic acid ends to the host DNA ends. Viral insertion often occurs at a specific locus or related loci in the host chromosome characteristic of the virus or viral family. A pathogenic provirus present in a target cell population may be associated with a specific condition or disease. Insertion of a provirus within a chromosomal exon or near an intron-exon border may lead to the production of an abnormal version of a normally transcribed mRNA and/or translated protein, with subsequent deleterious effects on the cell.
Chromosomal translocations are genetic recombination events associated with certain cancers, such as some leukemias and lymphomas. In some translocations, two different chromosomes reassemble by reciprocal genetic exchange between the chromosomes forming two hybrid chromosomes, each including portions of two normal chromosomes. Other translocations are intrachromosomal, joining normally noncontiguous regions of the same chromosome. A “breakpoint junction” of the translocation refers to a joining point of sequences derived from normally separated chromosomal locations. In some translocations, the breakpoint junctions are clustered in conserved locations within one or both of two chromosomes. Translocations occurring within a genetic coding region may result in atypical mRNA having discrete regions, such as a 5′ portion derived from one chromosomal location and a 3′ portion derived from another chromosomal location. Such novel transcription products are known as “fusion transcripts” or “fusion mRNA.”
One family of translocation events are characteristic of patents having chronic myelogenous leukemia (“CML”). The translocations occur between human chromosomes 9 and 22 (referred to as “t(9:22)”), and the resulting shortened chromosome 22 is known as the Philadelphia chromosome or Ph1. The t(9:22) events link portions of the abl gene of chromosome 9, and the “breakpoint cluster region” or bcr gene of chromosome 22. A cDNA prepared from an about 8 kb fusion mRNA isolated from a Ph1-positive cell line has been sequenced, revealing both abl and bcr sequences that code for a fusion protein having a tyrosine kinase activity (Shtivelman et al., Nature 315:550-554, 1985). The translocation occurred between the abl exon 2 and the bcr b3 region. Antibody-detection of an altered Abl protein having a molecular weight higher than that of normal Abl protein has been used for detecting Ph1-positive cells, diagnostic of CML, as described in U.S. Pat. No. 4,599,305 to Witte et al.
A subtype of acute lymphoblastic leukemia (ALL) is also characterized by a translocation between chromosomes 22 and 9, within a bcr region about 50 kb 5′ of the CML-associated bcr region. These ALL-associated translocations occur within the putative first intron of the bcr region, giving rise to a novel chimeric mRNA containing a splice between the bcr b1 of chromosome 22 and the abl exon 2 of chromosome 9, producing a novel fusion protein (Hermans et al., Cell 51:33-40, 1987). ALL-associated translocations on chromosome 22 have been detected with probes specific for a portion of the bcr gene as disclosed in European Pat. Publication No. EP 0 364 953 by Nakamura et al.
Chromosomal translocations involving chromosomes 8 and 21 have been associated with up to 40% of reported cases of pediatric acute myelogenous leukemia (“AML”) with the FAB-M2 morphology. The breakpoints on both chromosomes are variable, but generally result in a common fusion transcript containing 5′ portions of the AML1 gene of chromosome 21 and 3′ portions of the ETO gene of chromosome 8, which gives rise to a fusion protein (U.S. Pat. No. 5,580,727 to Ohki et al.).
Other chromosomal translocations associated with diseases such as lymphomas and leukemias are the t(15;17) translocation (ALL), and the t(12;21), t(4;11) and the t(1;19) translocations (AML).
The detection of chimeric DNA and/or RNA and/or fusion proteins associated with conditions and diseases such as those exemplified above would be useful in confirming initial diagnosis, in monitoring a patient's response to treatment, and in providing early warning of any recurrence of disease after a period of remission. One problem previously limiting the ability to detect chimeric nucleic acids or proteins has been the extremely small numbers of cells that are present at different stages of the condition, especially following remission or in a non-acute phase. Immunological techniques are not generally sensitive enough to detect such small amounts of analyte in a sample. The prognosis for patients having conditions associated with chromosomal translocations, including recurrence of the condition, is usually more favorable with early diagnosis compared to later diagnosis.
Methods for the diagnosis of conditions associated with chromosomal translocations, such as CML and ALL have been reported. U.S. Pat. No. 4,681,840 to Stephenson et al. and PCT Pub. No. WO 85/03297 disclose DNA and hybridization methods for direct detection of CML-associated Philadelphia chromosome abnormalities using a chromosomal DNA-derived probe containing sequences complementary to the bcr region. CML-associated t(9:22) translocations resulting from splicing events that join bcr b2 or b3 with abl exon 2 can be detected by amplifying the fusion mRNA and hybridizing thereto synthetic oligonucleotide probes specific for these spliced sequences, as described in U.S. Pat. No. 4,874,853 to Rossi et al. and European Pat. Pub. No. 0338713. Methods of detecting unique aberrant gene transcripts of a targeted genomic abnormality by hybridizing the RNA with one or more synthetic DNA oligonucleotides complementary to RNA sequences encoded by the Ph1 chromosome, thereby forming an RNA-DNA heteroduplex resistant to enzymatic degradation, followed by PCR amplification and DNA detection as an indication of the presence of unique aberrant gene transcripts are described in U.S. Pat. No. 4,999,290 to Lee. The DNA oligonucleotides include bcr b2 and/or b3 and abl sequences. DNA sequences for detecting and identifying chromosomal aberrations in tumor DNA containing the ALL-1 breakpoint region of human chromosome 11 (e.g., t(9;11 translocations) using probe hybridization in a variety of methods (Southern or Northern blotting or in situ hybridization) are disclosed in U.S. Pat. Nos. 5,567,586 and 5,633,136 to Croce et al.
Methods for detecting translocations using junction probes are limited to detecting mRNA or DNA resulting from specific fusion events because each detection probe is directed to a specific breakpoint junction. For translocations such as those associated with CML in which a large number of breakpoints can occur within a small area of the bcr gene, detection of mRNA resulting from each such fusion would require use of a different breakpoint junction probe. Also, point mutations that may limit probe hybridization are sometimes found within a few bases of a splice junction. Furthermore, other chromosomal rearrangement events such as deletions or insertions within the breakpoint cluster region may also produce less-common CML-associated transcripts. These events would result in mRNA or DNA species that would not be detected by using a common breakpoint junction probe.
U.S. Pat. No. 5,487,970 to Rowley et al. discloses methods for the detection of chromosome 11 (11q22) translocations, using a breakpoint probe such as mentioned above, or a method employing fluorescent in situ hybridization (FISH). In the FISH method, two fluorescently-labeled probes directed to 11 q22 chromosomal regions flanking a commonly-found breakpoint are hybridized to chromosomes in situ which are then are observed using a microscope illuminated at the light wavelength at which the fluors absorb. Cells in which the label is present only on chromosome 11 are classified as normal, whereas cells in which the label appears on different chromosomes are identified as possessing a translocation. This method requires a relatively large cell population to screen if the translocation is not present in many cells in the sample. The disclosed probes include restriction endonuclease fragments that can hybridize to chromosomal translocations involving the ALL-1 gene of chromosome 11 in a FISH or Southern blot format. Other methods for detecting CML-associated translocations using FISH involve probes derived from species-specific DNA regions between repeat segments (“inter-Alu” sequences) as disclosed in U.S. Pat. No. 5,538,869 to Siciliano et al.
The aforementioned methods rely on direct detection of the chimeric RNA or DNA by nucleic acid hybridization without signal or target amplification. Therefore, these detection methods require a relatively large number of the target cells bearing the chromosomal rearrangement in the tissue, blood, or other body fluid from which the sample is taken. However, large numbers of abnormal cells are not generally present in a biological sample when the disease appears to be in remission or is in a chronic non-acute state. Thus, by the time sufficient amounts of cells bearing genetic abnormalities are present to permit direct detection, a patient's treatment options may be limited.
Other methods of target nucleic acid detection use amplification to obtain multiple copies of a target nucleic acid (either or both of the complementary strands) or a reporter molecule to increase detection sensitivity. A variety of nucleic amplification methods are known to those skilled in the art (e.g., see Persing, D. H., “In Vitro Nucleic Acid Amplification Techniques” in Diagnostic Medical Microbiology: Principles and Applications 51 (Persing et al., Ed., 1993)).
Polymerase chain reaction (PCR) permits detection of small amounts of DNA present in a sample by amplifying the DNA target using two or more primers and a repeated series of thermal denaturation, primer annealing and synthesis steps (U.S. Pat. No. 4,683,195 to Mullis et al.; PCR Protocols: A Guide to Methods and Applications, Innes, M. A. et al., Ed., Academic Press, Inc., 1990, San Diego, Calif.). Generally, a first primer hybridizes to a specific region of the target nucleic acid, and a second primer hybridizes to the opposite strand of the target DNA 5′ to the binding site of the first primer to produce by enzymatic means a series of primer extension products that exponentially amplify the region between the primers.
The ligase chain reaction (LCR) uses two complementary sets of short DNA oligonucleotides that hybridize to adjacent regions of a target nucleic acid and the oligonucleotides are then covalently linked by using a DNA ligase (European Pat. No. 0320308). By employing repeated cycles of thermal denaturation, hybridization and ligation an accumulated double-stranded ligated oligonucleotide product can be detected, indicating the presence of the target nucleic acid in a sample.
Strand displacement amplification (Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396,1992), employs oligonucleotide primers that contain restriction endonuclease cleavage sites and hybridize to opposite strands of a target nucleic acid duplex (i.e., on the + and − sense strands) on either side of the sequence to be amplified. DNA polymerase-mediated primer extension using three deoxynucleoside triphosphates (dNTP) and a single dNTP[α]S produces a duplex hemiphosphorothioated primer extension product, which is nicked rather than cut by restriction endonuclease. Then, the 3′ end of the nick is extended by a DNA polymerase that simultaneously displaces the non-hemiphosphorothioated strand. Each displaced strand of one sense can serve as a template for the binding of oligonucleotide primers of the opposite sense, resulting in the geometric accumulation of double stranded nucleic acids.
Another nucleic acid amplification method employs an RNA replicase (Qβ replicase) capable of amplifying the probe itself (e.g., see U.S. Pat. No. 5,112,734 to Kramer et al.)
Transcription based amplification systems employ an RNA polymerase to make RNA transcripts of a target region (see U.S. Pat. Nos. 5,480,784 and 5,399,491 to Kacian & Fultz). One method, termed transcription-mediated amplification (TMA), uses a promoter-primer that hybridizes to a target nucleic acid in the presence of a reverse transcriptase and an RNA polymerase to form a double stranded promoter. Then, RNA transcripts are produced which become templates for further rounds of TMA in the presence of a second primer capable of hybridizing to the RNA transcripts. Unlike PCR and other methods that require heat denaturation, TMA is an isothermal amplification method that uses an RNAse H activity to digest the RNA strand of an RNA-DNA hybrid, thereby making the DNA strand available for hybridization with a primer or promoter-primer. Generally, the RNAse H activity is supplied by a retroviral reverse transcriptase provided for amplification.
Chromosomal translocations and their transcription products have been detected using methods that include nucleic acid amplification. A PCR-based method to detect cells containing genomic DNA having t(14q32;1 Bq21) chromosomal translocations associated with follicular lymphomas, and especially with minimal residual or relapse disease, by amplifying sequences surrounding a breakpoint cluster region, have been described in U.S. Pat. No. 5,024,934 to Lee. PCR primers and cDNA amplification methods for identifying t(9;11) translocations in a patient's tissue have been described in U.S. Pat. No. 5,633,135 to Croce et al. Methods for detecting carcinoma metastases (one in 10,000 to 100,000 cells) involving PCR amplification of target nucleic acids have been described by Green et al. in European Pat. Publication No. EP 520794.
Similarly, PCR amplification has been used to detect chimeric mRNA associated with ALL (U.S. Pat. No. 5,057,410 to Kawasaki et al.) and RNA transcripts originating from t(8;21) translocation in leukemic cells (U.S. Pat. No. 5,547,838 to Nisson et al.). U.S. Pat. No. 5,057,410 to Kawasaki et at discloses PCR methods to detect chimeric mRNA containing exon—exon junctions. In this method, mRNA extracted from the target cell is reverse transcribed to produce cDNA that is amplified by PCR to make a double-stranded DNA amplification product. This amplification product is then denatured and one of the resulting strands is detected by hybridizing an oligonucleotide probe to the breakpoint junction. Individual probes hybridize to distinct bcr-abl junction species associated with CML and ALL.
Sooknanan at al. (Experimental Hematol. 21:1719-1724,1993) described a method for the detection of bcr abl mRNA characteristic of CML by extracting RNA from FICOLL™-fractionated peripheral blood leukocytes and amplifying a target nucleic acid using an isothermal amplification procedure involving transcription. Serial reactions using four amplification primers (a primary pair in the first reaction and a nested pair in the second reaction) were essential for amplification and detection of bcr-abl mRNA. The amplified nucleic acid produced by this method was of the same sense as the original mRNA and was detected using two different bcr-abl junction probes for detecting two splice junctions. Probes to the breakpoint junctions were used to distinguish normal bcr or abl mRNA from the chimeric bcr-abl mRNA in a patient sample.
PCR-based methods have been used for detecting other translocations associated with cancers or their transcription products. Probes and PCR primers for detecting t(2;13) translocations associated with alveolar rhabdomyosarcoma have been described in U.S. Pat. No. 5,650,278 to Barr et al. A nucleic acid sequence encoding a fusion protein (anaplastic lymphoma kinase or ALK) associated with t(2;5) lymphomas has been disclosed in U.S. Pat. No. 5,529,925 to Morris et al.
There remains a need in the art for a simple, sensitive, and rapid method for the detection of chimeric nucleic acids, particularly chimeric mRNA obtained from biological samples such as blood, marrow, plasma, biopsy tissue, sputum, urine, feces, semen or other body fluids. Preferably, such methods would require a minimum of technical expertise by laboratory personnel and a minimum of specialized laboratory equipment such as centrifuges, thermocycling and electrophoresis equipment, and would use relatively low-cost reagents. Furthermore, there exists a need for an assay for detecting chimeric mRNA that would provide a result capable of interpretation with a minimum of qualification by, for example, reducing the possibilities of false positive and false negative results.
There also exists a need in the art for a simple and rapid method of preparing cytoplasmic nucleic acids, particularly mRNA, for use as a potential target in diagnostic nucleic acid hybridization assays or as a template for nucleic acid amplification. Such simplified preparative methods reduce the need for exhaustive extraction and purification procedures.
A crucial element of methods of detecting RNA (or amplification products made therefrom), particularly mRNA, is the manner of extracting RNA from the cells. Because of the ubiquitous presence of various RNases, extraction methods must preserve the small amounts of target RNA that may be present in a sample. Extraction methods that liberate all nucleic acids (including nuclear nucleic acids) from the target cell, produce samples that contain not only of the target mRNA but also the DNA encoding it which can produce false positive results in many nucleic acid assays.
Previously reported extraction techniques generally involve the use of a chaotropic agent such as guanidinium to lyse the cells. Further processing has typically involved mechanical shearing of the DNA, phenol and chloroform extraction, and ethanol precipitation, or LiCl precipitation of the RNA from the guanidinium. Additional methods specific for mRNA preparation have employed oligo dT (polythymine) immobilized on resins to capture polyadenylated mRNA.
It would be advantageous in diagnostic methods that detect mature cytoplasmic RNA to be able to permeabilize cells to release mature RNA species into the extraction buffer, while not appreciably releasing nuclear material. Thus, DNA and immature nuclear RNA species would not be mixed with the desired target nucleic acid. By precluding initial mixing of desired RNA species and contaminants that contain the same or complementary sequences and/or increase viscosity, additional steps to eliminate iv chromosomal DNA and false positive results may be avoided. A rapid, simple lysis method that will liberate cytoplasmic RNA species while not significantly releasing pre-mRNA or DNA, and that does not require a high level of specialized skill or training is particularly desirable for use in commercial assays and kits. There exists a need for a rapid, easy lysis method that yields RNA suitable for qualitative and/or quantitative nucleic acid amplification or direct detection of specific nucleic acids.